PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease. For the majority of patients chemotherapy is the only therapeutic option, and virtually all patients relapse with drug resistant disease. The nucleoside analogue gemcitabine is the frontline agent and current standard of care for patients with PDAC. When combined with gemcitabine, erlotinib or nab-paclitaxel increases median survival of patients with non- resectable disease by ~0.3 to ~1.8 months, respectively. The five-year survival is ~6%. Clearly more effective therapies are needed. We propose to develop effective therapy for patients with PDAC. Our data demonstrate that the combinations of gemcitabine + the BET (bromodomain extra-terminal domain) inhibitor JQ1 and also of JQ1 + the PARP 1/2 inhibitor olaparib induce strongly synergistic cytotoxicity in PDAC cells in vitro. Notably, a pilot study with JQ1 + gemcitabine supports in vitro data, and shows that this combination induces regressions in mice bearing patient-derived xenograft (PDX) tumors. The synergy of both combinations that we propose to evaluate was evident in PDAC cells that express mutant KRAS, a tumor cell characteristic associated with poor outcome clinically. The data suggest that JQ1 + gemcitabine and JQ1 + olaparib may comprise effective therapy for PDAC. JQ1 is a novel targeted small molecule that binds to acetyl lysine binding pockets of BET proteins, predominantly BRD2 and BRD4. This binding inhibits the association of BRD2/4-dependent transcriptional aggregates to acetylated lysine residues of histones, thereby inhibiting expression of proteins dependent on this mechanism. Data in this proposal show the novel findings that JQ1 induces DNA damage, inhibits expression of the G2 cell cycle regulator protein CDC25B and DNA repair proteins Ku80 and BRCA2 in vivo. We hypothesize, based on our findings and relevant literature, that JQ1-induced DNA damage, inhibition of CDC25B and consequent cell cycle dysregulation sensitize PDAC cells to gemcitabine. We also hypothesize, based on our findings and relevant literature, that JQ1-induced inhibition of Ku80 and BRCA2 and consequent suppression of DNA repair sensitizes PDAC tumors to PARP inhibitors. We propose to evaluate the efficacy and mechanisms of synergy of JQ1 + gemcitabine (Aim 1) and JQ1 + olaparib (Aim 2) using transfected PDX- derived cell lines in vitro and our unique panel of in vivo PDX models that retain specific biologic and genetic characteristics of their tumors of origin. We also propose to identify additional proteins that contribute to the cytotoxic mechanism of JQ1 (Aim 3). The proposed work comprises an essential step toward our long-range goal of developing effective therapy for patients with PDAC.